RBS said uncertainty could affect its own credit ratings as well as change the wider business environment.
Still 80 per cent owned by the state, the bank, which says it is neutral ahead of the vote, made a similar comment earlier this year in its annual report.
Since then it has been holding talks with the Bank of England and the Scottish and UK Governments over the referendum.
Ian Murray, Labour's shadow business minister, said: "That RBS have reiterated their concerns about separation underlines the threat it poses to Scottish jobs, savings and mortgages."
Scottish Conservative finance spokesman Gavin Brown said the comments were "another example of a financial services key player raising legitimate worries about the impact of independence".
"All of these institutions can't be wrong, and it's time the SNP offered some kind of credible reassurance about the future of major headquarters and jobs in the event of independence."
In response a Scottish Government spokesman said: "This is simply a restatement of the position set out in RBS annual report which they are legally obliged to make and puts the referendum alongside UK interest rates, the housing bubble and ongoing banking reforms as issues they are monitoring.
"As (RBS boss) Ross McEwan himself pointed out earlier this year, RBS already operates in 38 countries around the world, and if it needed to be 39 then he said 'that's what we'll do'."
He added an independent Scotland would use Sterling as part of formal "arrangements that would ensure both currency and regulation remained the same across the UK", a claim disputed by the Coalition Government.